PROJECT SUMMARY Lyme disease is the most prevalent arthropod-borne disease in the US. The causative agent of the disease is the bacterium Borrelia burgdorferi (Bb), which affects multiple tissues including the central nervous system (CNS). Lingering symptoms including pain, fatigue, and difficulties with memory and cognition persist in up to 20% of all patients treated with antibiotics. The cause of this Post-Treatment Lyme Disease Syndrome (PTLDS) is unclear and controversial. We propose that live bacteria are not required to drive persistent inflammation in the CNS. Using a rat model of neuroborreliosis, we demonstrated that CNS inflammation persists in the absence of live bacteria. What drives the persistent inflammation? We also found Bb can induce changes in microRNA expression changes in astrocytes including miR-122-5p, miR-135a-3p, miR-135a-5p, miR-143-3p and miR-146b-5p. Targets of these microRNAs include genes involved in cell adhesion, tight junctions, cell signaling, and response to infection. Astrocytes are key players in response to injury, neuronal support, and tissue repair. Our working hypothesis is that neurological symptoms are perpetuated by Bb-induced microRNAs that lead to a persistent neuroinflammatory response. We will test this hypothesis with 2 Specific Aims. In Specific Aim 1 we will develop a complete atlas of the glial- specific microRNAs and their targets induced Bb-infected brain relative to uninfected brain. In the second Specific Aim, we will manipulate microRNA-mediated inflammatory mediators in primary glial cells through the use of specific microRNA antagomirs, demonstrating the mechanistic link between altered microRNA expression and inflammation. Our long-term objective is to modulate host CNS responses to Bb in vivo through manipulation of microRNA expression. By elucidating the specific microRNAs that contribute to neuroinflammation, we will provide novel targets for Lyme disease therapy. MicroRNA mimics and antagonists are already in human trials, demonstrating the potential feasibility of such therapies.